Molecular cloning and phylogenetic characterisation of beta-2 microglobulin in canine mammary tumours

Introduction

Cancer remains one of the leading causes of morbidity and mortality worldwide, posing a major public health challenge. According to recent global estimates, approximately 19.3 million new cancer cases and nearly 10 million cancer-related deaths were reported in 2020, with the burden expected to rise to over 28 million new cases annually by 2040 due to population growth, aging, and lifestyle changes (Sung et al., 2021; Ferlay et al., 2021) [14,3] . Among all cancer types, breast, lung, colorectal, and prostate cancers account for a significant proportion of global incidence and mortality. In addition, disparities in cancer burden are evident between developed and developing countries, where limited access to early diagnosis and treatment contributes to poorer outcomes (Bray et al., 2018) [2] . β2-Microglobulin (B2M) is a low molecular weight (~12 kDa), non-glycosylated protein that constitutes the light chain of major histocompatibility complex class I (MHC-I) molecules and is expressed on the surface of all nucleated cells. It plays a crucial role in antigen presentation and immune surveillance by stabilising MHC-I complexes and facilitating the recognition of intracellular antigens by CD8⁺ T lymphocytes (Jackson et al., 1999; Ploegh, 1988) [7,9] . Under normal physiological conditions, B2M is continuously shed into circulation, and its serum concentration reflects cellular turnover and immune system activity, making it a useful biomarker in various pathological conditions, including cancer (Prizment et al., 2016) [10] . In human cancers, B2M has been extensively studied as both a diagnostic and prognostic biomarker. Elevated levels of circulating B2M have been reported in haematological malignancies such as multiple myeloma, lymphoma, and leukaemia, as well as in solid tumours including breast, lung, prostate, and renal cancers. Increased B2M levels are strongly associated with tumour burden, disease progression, and poor clinical outcomes (Bataille & Klein, 1992; Zhang et al., 2013) [1,15] . In addition to its role as a biomarker, B2M actively contributes to tumour progression by modulating signalling pathways involved in proliferation, apoptosis, angiogenesis, and metastasis. For instance, B2M has been shown to activate pathways such as protein kinase A/CREB and PI3K/Akt, thereby promoting cancer cell survival and growth (Huang et al., 2006) [6] . Conversely, loss or mutation of B2M can impair MHC-I expression, leading to reduced antigen presentation and enabling tumour immune evasion, which contributes to resistance to immunotherapy (Han et al., 2025; Reis et al., 2024) [5,12] . Comparative oncology offers a valuable approach to understanding cancer biology across species, particularly between dogs and humans. Canine cancers develop spontaneously and share significant similarities with human malignancies in terms of histopathology, molecular pathways, tumour microenvironment, and clinical progression. Among these, canine mammary tumours (CMTs) are one of the most common neoplasms in female dogs and closely resemble human breast cancer, making them an important translational model (Queiroga & Lopes, 2011; Nosalova et al., 2024) [11,8] . [4,13] .

Materials and Methods

Immunohistochemistry for β2-microglobulin

Paraffin-embedded tissue sections (4-5 µm) were mounted on poly-L-lysine (Sigma-Aldrich) coated slides and incubated at 60 °C for 1 h. Sections were deparaffinized in xylene (Merk) and rehydrated through graded ethanol to distilled water. Antigen retrieval was performed in a citrate buffer (10 mM, pH 6.0; Abcam) in a microwave oven for 10-15 min, then cooled at room temperature. Endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide (Sigma-Aldrich) for 10 min. Nonspecific binding was minimised by incubating the sections with normal serum or blocking buffer for 20 min. The sections were then incubated overnight at 4 °C with a polyclonal anti-β2-M antibody (1:50; Thermo Fisher Scientific, PA5-87248; in 2.5% BSA). After washing with PBS, sections were incubated with HRP-conjugated antidog secondary antibody (Sigma-Aldrich, 1:200) for 60 min at room temperature. Immunoreactivity was visualised using 3,3′-diaminobenzidine (DAB) as the chromogen, resulting in a brown precipitate at antigen sites. Sections were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted with DPX.

RNA isolation and cDNA synthesis

Total RNA was extracted from tumour and control tissue samples using TRIzol® reagent (Invitrogen, USA) following the manufacturer's protocol. The concentration and purity of isolated RNA were determined using a NanoDrop™ spectrophotometer (Thermo-Fisher Scientific, USA) by measuring absorbance at 260/280 nm. RNA integrity was confirmed by electrophoresis on a 1.5% agarose gel. For complementary DNA (cDNA) synthesis, 1 µg of total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermoscientific) in a final reaction volume of 20 µL, according to the manufacturer's instructions. The synthesised cDNA was stored at -20 °C until further analysis.

PCR amplification of the B2M Gene

The canine Beta-2 microglobulin (B2M) gene was amplified using gene-specific primers designed from the conserved canine B2M coding sequence. Primer design was performed using Primer Premier 5.0 (National Bioscience) and OligoAnalyzer tool 3.1 (Integrated DNA Technologies, USA). PCR amplification was carried out in a thermal cycler using synthesised canine mammary tumour-derived cDNA as template. The reaction mixture consisted of template cDNA, gene-specific forward and reverse primers, Phusion polymerase (Thermo-Scientific), dNTPs (Takara) and nuclease-free water in optimised concentrations. Amplified PCR products were resolved on 1.5% agarose gel electrophoresis and visualised under ultraviolet illumination following nucleic acid staining.

Cloning of the canine Β2M gene

The 50 µL PCR-amplified B2M gene products were purified using the MinElute PCR Purification Kit (Qiagen). Purified amplicons were subsequently digested with KpnI and SalI restriction enzymes (New England Biolabs, USA). Following restriction digestion, the digested products were further purified using a PCR cleanup kit (Qiagen) to remove residual enzymes and reaction components. The purified inserts were then ligated into the pH6HTN His₆ HaloTag PET32 expression vector (Promega) using T4 DNA ligase (Takara Bio Inc., Japan). The recombinant plasmid constructs were transformed into Escherichia coli DH5α competent cells and plated onto Luria-Bertani (LB) agar plates supplemented with 100 µg/mL ampicillin for selection of positive transformants.

Phylogenetic analysis

Identification of nucleotide sequences homologous to the isolated B2M gene was performed using BLAST against the NCBI nucleotide database. The retrieved B2M sequences were aligned using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) algorithm implemented in MEGA 7 software (REF). Phylogenetic relationships were subsequently inferred using the maximum-likelihood method after selection of the best-fit nucleotide substitution model.

Results

Clinical characteristics and sample collection of canine tumour cases

Gross examination of canine mammary tumour cases revealed pronounced heterogeneity in tumour size, shape, and external morphology. The excised tumour mass (Fig. 1A) was typically irregular, firm, and lobulated, with focal areas of congestion and haemorrhage, consistent with active and progressive tumour growth. vernier calliper-based measurements demonstrated substantial tumour dimensions, indicative of advanced tumour development. Preoperative in vivo assessment of the affected mammary gland (Fig. 1B).

Histopathological classification of canine mammary tumours

Histopathological examination of canine mammary tumour sections revealed diverse morphological patterns corresponding to different tumour subtypes. Features of adenosquamous carcinoma, characterized by mixed glandular and squamous differentiation with marked cellular pleomorphism and prominent nucleoli (Fig. 2A). Demonstrated carcinoma in situ, with neoplastic epithelial cells proliferating within well-defined ductal structures, remaining confined by an intact basement membrane, indicating a non-invasive stage of tumour progression (Fig. 2B). Features of comedocarcinoma were observed, including central areas of necrosis within dilated ducts surrounded by malignant epithelial cells, suggestive of aggressive tumour behaviour (Fig. 2C). It exhibited characteristics of complex-type carcinoma, with the presence of both epithelial and myoepithelial components arranged in irregular nests and cords, indicating biphasic tumour morphology (Fig. 2D). It revealed fibrosarcoma, composed of spindle-shaped neoplastic cells arranged in interlacing bundles within a collagen-rich stroma, reflecting mesenchymal tumour differentiation (Fig. 2E). Normal canine mammary gland tissue exhibiting preserved ductal and alveolar architecture with normal epithelial cell morphology, included as a non-neoplastic control for comparative pathological and molecular analyses (Fig. 2F).

Immunohistochemical expression of β2microglobulin

Immunohistochemical analysis demonstrated distinct β2microglobulin expression in canine mammary tumour tissues. Representative tumour sections showed positive immunoreactivity characterized by brown chromogenic staining predominantly localized to the cytoplasm and cell membrane of neoplastic mammary epithelial cells. The intensity of staining ranged from moderate to strong and was more pronounced in densely cellular and poorly differentiated tumour regions, indicating elevated B2M expression in malignant tissues (Fig. 3A-B). The negative control section (Fig. 3C), processed without the primary antibody, exhibited no specific staining, confirming the specificity of the immunohistochemical reaction.

PCR amplification of canine B2M gene

Successful amplification of the canine Beta-2 microglobulin (B2M) gene was achieved from canine mammary tumourderived cDNA samples using gene-specific primers. Agarose gel electrophoresis analysis revealed distinct and well-defined amplification bands corresponding to the expected amplicon size of 288 bp in lanes L1-L4, confirming efficient amplification of the target B2M gene across all analysed tumour samples (Fig. 4). The amplified B2M PCR products were visualised under ultraviolet illumination following electrophoretic separation on an ethidium bromide-stained 1.5% agarose gel.

Cloning and molecular confirmation of the canine B2M gene

The PCR-amplified canine B2M gene was successfully cloned into the pET-32b expression vector and transformed into Escherichia coli DH5α competent cells. Numerous bacterial colonies were observed on LB agar plates supplemented with ampicillin, indicating successful transformation and uptake of the recombinant plasmid construct (Fig. 5A). Restriction digestion analysis of the recombinant plasmid revealed the presence of the linearized pET-32b vector of approximately 5900 bp along with the released B2M insert of 288 bp, confirming successful ligation of the target gene into the expression vector (Fig. 5B). Furthermore, colony PCR screening of transformed DH5α colonies generated distinct amplification bands corresponding to the expected 288 bp B2M gene fragment (Fig. 5C), thereby confirming the presence of the recombinant construct in positive bacterial colonies. These confirmed recombinant clones were subsequently utilized for downstream molecular characterization and expression studies.

Phylogenetic analysis of the B2M gene

The phylogenetic analysis of the β2-microglobulin (B2M) gene revealed clear evolutionary relationships among the analyzed sequences. The B2M sequences clustered into multiple clades, indicating a high degree of conservation with moderate sequence divergence across species. Closely related sequences grouped together within the same clades, supported by varying bootstrap values, confirming the reliability of the observed clustering patterns. The Canis lupus familiaris B2M sequence clustered with homologous mammalian B2M sequences, demonstrating its close evolutionary relationship with other carnivores and higher mammals. Several internal nodes showed moderate to high bootstrap support (≥70%), suggesting stable evolutionary lineages, whereas some branches exhibited lower bootstrap values, reflecting minor sequence variations and possible evolutionary divergence. Overall, the phylogenetic tree indicates that the B2M gene is evolutionarily conserved, with species-specific variations contributing to distinct sub-clustering. These findings support the functional conservation of B2M across species and validate its suitability for comparative and evolutionary studies, as well as for translational relevance between canine and other mammalian cancer models (Fig. 6).

Discussion

The present study provides a comprehensive evaluation of β2-microglobulin (B2M) in canine mammary tumours (CMTs) through histopathological, immunohistochemical, and molecular approaches, highlighting its potential role as a biomarker and its relevance in comparative oncology. The observed heterogeneity in tumour morphology and size is consistent with previous reports describing the diverse biological behaviour of CMTs, ranging from benign to highly aggressive malignant phenotypes (Queiroga & Lopes, 2011; Sorenmo et al., 2009) [11,13] . Histopathological analysis revealed that the majority of tumours were malignant, predominantly classified as tubular, tubulopapillary, complex, and solid carcinomas with moderate to poor differentiation (grades II-III). These findings align with earlier studies indicating that highergrade tumours exhibit increased cellular atypia, mitotic activity, and stromal invasion, which are hallmarks of aggressive tumour behaviour and poor prognosis (Goldschmidt et al., 2011; Nosalova et al., 2024) [4,8] .

The successful amplification and cloning of the canine B2M gene further validate the molecular integrity and conserved nature of this gene. The identification of a distinct 288 bp amplicon confirmed the specificity and reliability of the experimental approach employed in the present study. Similar molecular characteristics and conserved expression patterns of B2M have been reported across different species, supporting its fundamental biological role in cellular and immune regulatory mechanisms (Jackson et al., 1999) [7] . The phylogenetic analysis demonstrates a high degree of evolutionary conservation of the β2-microglobulin (B2M) gene across species, indicating its essential and conserved biological function in MHC-I-mediated antigen presentation (Jackson et al., 1999) [7] . This conserved functional role is critical for maintaining immune surveillance and cellular homeostasis. Importantly, the preserved structure and function of B2M across species suggest that its dysregulation may have similar pathological consequences, including tumour progression and immune evasion. In the context of cancer, elevated B2M expression has been linked to activation of oncogenic signalling pathways and resistance to immune-mediated tumour clearance (Huang et al., 2006; Han et al., 2025; Reis et al., 2024) [6,5,12] .

Conclusion

β2-Microglobulin (B2M) is significantly associated with tumour progression and higher histological grades and immunohistochemical analysis in canine mammary tumours, indicating its potential as a diagnostic and prognostic biomarker. Its elevated expression in malignant tissues and conserved molecular characteristics highlight its functional role in tumour biology. Phylogenetic analysis further supports its evolutionary conservation and translational relevance. These findings suggest that B2M may serve as a promising target for future cancer diagnostics and therapeutic strategies.

Acknowledgement

AcknowledgementsThe authors are grateful to the Director, ICAR-IVRI, Izatnagar, for providing the necessary funding and support. The study was conducted with support from Grants received under ICAR LAL Bahadur Shastry Outstanding Young Scientist Award Project, (F.no 5-4/2022-A.C (e.21930) and Advanced Research Project on Canines.

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